Electrochemiluminescent measurement techniques derive from electrochemistry and chemiluminescent detection techniques. Electrochemistry (EC) deals generally with the relation of electricity to chemical changes and with the interconversion of chemical and electrical energy. Chemiluminescence (CL) based assay or detection techniques include, for example, binding assay techniques which generally comprise forming a mixture of a sample containing an unknown amount of an analyte of interest to be determined with a known amount of a reactant which is conjugated with a chemiluminescent label. The mixture is incubated to allow the labeled reactant to bind to the analyte. After incubation, the mixture is separated into two fractions: a bound and an unbound fraction. The bound fraction is labeled reactant bound to analyte and the unbound fraction is the remaining unbound reactant. The CL measurement is then taken. The fractions are chemically caused to luminesce, for example by the addition of an oxidant to the fractions. The bound and unbound fractions of the labeled reactant will emit different amounts of light. The measured level of chemiluminescence at the specific wavelength is indicative of the amount of the bound and/or unbound fraction, respectively and from such measurements one skilled in the art can determine the amount of analyte in the sample.
Electrochemiluminescent (ECL) detection techniques provide a sensitive and controllable measurement of the presence and amount of an analyte of interest. In ECL techniques, the incubated sample is exposed to a voltammetric working electrode, that is, an electrode to which a voltage is applied and from which a current for a redox reaction may be passed. The ECL mixture does not react with the chemical environment alone, as does the CL mixture, nor with an electric field alone as in EC, but rather electrochemiluminescence is triggered by a voltage impressed on the working electrode at a particular time and in a particular manner to controllably cause the ECL moiety to emit light at the electrochemiluminescent wavelength of interest. The voltage impressed on the working electrode generates the oxidant in situ which, in the CL methodology, is added externally.
The reverse side of this sensitivity is that, in general, a particular sample of the reactive mixture may not be measurable twice to produce exactly the same result, although sometimes the results may be close enough to be considered the same. The ECL measurement is destructive, in that the sample changes its chemical composition during the measurement. In accordance with a proposal by employees of the assignee of the present application who are under an obligation of assignment to the present assignee, the working electrode may be conditioned to provide a precisely controlled surface for subsequent ECL measurements. This provides controllable initial conditions for each individual sample in succession, but the conditions change after measurement.
It has further been proposed by another employee of the present assignee also under an obligation of assignment thereto to provide an internal standard ECL measurement, in which a known amount of labeled reactant is present in the reactive mixture to provide a calibrating signal against which to measure the electrochemiluminescence of the analyte of interest. The internal standard is therefore a second analyte of interest which can emit light at a second, different wavelength. Advantageously the internal standard will have the same chemical mechanism for chemiluminescence as the prime analyte of interest and differ only in the wavelength of its emitted light. Alternatively, a second analyte of interest which is not an internal standard may be present in the reactive mixture. Since the second analyte also emits light when triggered by the voltage impressed on the working electrode, its electrochemiluminescence will also be affected by the irreversible chemical changes during the ECL measurement process. It would be highly advantageous to measure the levels of electrochemiluminescence of the prime analyte of interest and the internal standard (or second analyte of interest) simultaneously before the response of either one is destroyed or reduced during the ECL measurement of the other.
It is known in some CL light measurements (which do not use electrodes for triggering a reaction) to measure light intensities at two different wavelengths for the same analyte to provide an intrinsic improvement in precision since some variations in the signal cancel. Two separate phototubes may be used for this purpose, with an appropriate filter for each, or a single phototube with rotating filters may alternatively be used. In the case of using two phototubes, there is no restriction in the spatial placement of these phototubes since the light is generated in the bulk of the CL solution, with the result that it is emitted uniformly in all directions.
The problem faced in ECL techniques, as discussed more fully below, is a different problem altogether which relates to the fact that light is produced as a result of chemical changes happening in a layer of sample immediately surrounding the electrode surface, due to the electrical potential imposed on the electrode. A technique which would preserve the chemical integrity of this layer for the two measurements would provide a more accurate measurement of the concentration of the two analytes.
Another difficulty in the measurement of light at two electrochemiluminescent wavelengths arises from the fact that the electrode by its nature prevents light from being emitted uniformly in all directions. This imposes geometric restrictions on the method to be used for the simultaneous measurement of the light that originates from the primary analyte and the light that originates from the internal standard.